Mmlv reverse transcriptase

Real-time RT-PCR was performed to measure the cyclin D1 mRNA levels in HCC cells as described previously [39 (link)]. Briefly, total RNA was isolated from the HCC cells using a RNeasy mini kit (Qiagen) according to the manufacturer's instructions, and reversely transcribed to cDNA using MMLV reverse transcriptase and the oligo(dT) primer kit (Solgent). Actin was used as the control gene. Real-time PCR was performed using SYBR Green I (Takara) and a Light-Cycler rapid thermal cycler (Roche Diagnostics). The primer sequences are shown in Supplementary Table 3. The relative VRK1 levels in HCC cells were determined using the 2^−ΔΔC_T method, as described [40 (link)].

BMMs were cultured with or without Ewha-18278 for the indicated periods. Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. After denaturation of total RNA at 70 °C for 10 minutes, first-strand cDNA was synthesized with oligo (dT) primers and MMLV-reverse transcriptase (SolGent, Seoul, Korea). The relative mRNA levels were evaluated by real-time PCR using a SYBR Green Master kit (Kapa Biosystems, Woburn, MA, USA) and reactions were performed in triplicate on ABI PRISM 7300 unit (Applied Biosystems, Franklin Lakes, NJ, USA). PCR primers were: NFATc1 sense, 5′-CCAGAAAATAACATGCGAGCC-3′; antisense, 5′-GTGGGATGTGAACTCGGAAG-3′; Atp6v0d2 sense, 5′-CAGAGATGGAAGCTGTCAACATTG-3′; antisense, 5′-TGCCAAATGAGTTCAGAGTG-3′; β-actin sense, 5′-ACCCTAAGGCCAACCGTG-3′; antisense, 5′-GCCTGGATGGCTACGTAC-3′. Melting curve analysis and agarose gel electrophoresis were performed to ensure a single PCR product. The relative expression levels were normalized to the level of actin mRNA as an internal control gene.

Total RNA was extracted from BMMs by TRIzol reagent (Invitrogen) and first-strand cDNA was synthesized with oligo (dT) primers and M-MLV reverse transcriptase (SolGent, Seoul, Korea). The relative mRNA levels were evaluated by quantitative RT-PCR (qRT-PCR) using SYBR Green Master kit (Kapa Biosystems, Woburn MA, USA). Gene specific primer sequences are provided in Supplementary Methods.